1. Field of the Invention
This invention relates to diagnosis of heart disease, and specifically relates to diagnosis of cardiac ischemia by detecting levels of free fatty acids in serum using a fluorescently-modified free fatty acid binding protein.
2. Description of the Prior Art
Ischemic heart disease affects millions of people worldwide, often leading to sudden death by acute myocardial infarction. Cardiac ischemia is often associated with chest pain (angina pectoris), generally caused by atherosclerosis, but asymptomatic individuals can also be at high risk because of hypertension, high serum cholesterol levels or family history. Myocardial ischemia occurs when blood flow to the heart is restricted or oxygen to heart muscle is compromised (hypoxia). Ischemia and hypoxia can lead to myocardial infarction, during which cardiac tissue is damaged resulting in abnormal cardiac muscle metabolism and contractions.
Diagnostic procedures for heart disease often assess the extent of cardiac tissue damage after symptoms are detected. Then, the disease may have progressed to an extent where AMI is imminent or has occurred. Moreover, about 25% of myocardial infarction (MI) patients display atypical symptoms resulting in misdiagnosis and discharge of about 5% of MI patients (Mair J. et al., Clin. Chem. 41:1266-1272, 1995; Newby L. K. et al., Clin. Chem. 41:1203-1265, 1995). Electrocardiography (ECG) monitoring of patients for MI detects the condition in only about half of the patients (Mair J. et al., Clin. Chem. 41:1266-1272, 1995).
ECG and currently available diagnostic blood tests are often not effective for detecting ischemia because they are designed to monitor infarction-associated tissue damage. Angina symptoms are often confirmed by ECG monitoring during treadmill exercise stress when the patient seeks treatment, but the test has a false negative rate of about 15%. Furthermore, exercise stress testing is usually too expensive and time consuming to be used to screen asymptomatic patients. Thus, a sensitive and reliable diagnostic test is needed for diagnosis of cardiac ischemia, especially for high-risk individuals.
Long-chain free fatty acids (FFA) are essential to normal physiological functions (e.g., as energy sources and cellular activity modulators). Levels of FFA in the serum that exceed the normal range found in serum of healthy individuals are indicative of certain patho-physiological states (Richieri, G. V. and Kleinfeld, A. M., J. Lipid Res., 1995, 36:229-240; Kleinfeld, A. M. et al., 1996, Am. J. Cardiol., 78:1350-1354). For example, impaired fatty acid metabolism occurs in individuals with diabetes mellitus, hyperlipidemia and associated with acute myocardial ischemia and/or infarction (Tekeishi, Y. et al., Nucl. Med. Commun., 1996, 17(8):675-680; Opie, L. H., Am. J. Cardiol. 1975, 36:938-953; Katz, A. M. et al., Circulation, 1981, 48:1-16; Hochachka, P. W., Science, 1986, 231:234-241; Oliver, M. F. and Opie, L. H., Lancet, 1994, 343:155-158). Myocardial FFA metabolism has also been used to evaluate cardiac function, for example, in patients with hypertensive-diabetic cardiomyopathy, cyanotic congenital heart disease and after coronary thrombolysis (Shimonagata, T. et al., Diabetes Care, 1998, 19(8):887-891; Kondo, C. et al., J. Nucl. Cardiol., 1996, 3(1):30-36; Franken, P. R. et al., 1994, J. Nucl. Med. 35(11):1758-1765). Typically, tomography imaging has been used to assess myocardial viablity and cardiac disease conditions by monitoring myocardial flow tracers (e.g., thallium-201) and uptake of fatty acid analogs (e.g., 123I-xcex2-methylliodophenylpentadecanoic acid (BMIPP))(reviewed in Franken, P. R. et al., Acta Cardiol., 1996, 51(6):501-514).
Amounts of cholesterol, FFA and lipoproteins in serum have been used as indicators of risk of heart disease. Amounts of phospholipids in cardiac muscle after myocardial infarct can be determined indirectly by measuring blood levels of cholesterol, FFA and xcex2-lipoproteins and then using a mathematical formula to calculate the phospholipid content (Soviet Union Pat. No. 1,270,705). FFA content was measured photo-electro-colorimetrically by forming insoluble complexes with a copper reagent.
Serum FFA is mostly bound to albumin but a significant minority is unbound (FFAU) and soluble in the aqueous phase. Concentrations of FFAU can measured using a method in which a fluorescently-labeled fatty acid binding protein (FABP) binds to the FFAU and thereby exhibits a fluorescence different from that exhibited when no FFA is bound (U.S. Pat. No. 5,470,714; PCT Pat. App. WO 91/09310; PCT Pat. App. No. WO 84/08014). The concentration of FFAU is obtained from the measured fluorescence difference. One such fluorescently-labeled fatty acid binding protein suitable for measuring FFAU concentrations is rat intestinal FABP derivatized at the Lys-27 residue with acrylodan, referred to as ADIFAB. Using ADIFAB and this method, it was shown that FFAU levels in serum samples from healthy donors were tightly regulated having a mean value of 7.5xc2x12.5 nM (Richieri, G. V. and Kleinfeld, A. M., J. Lipid Res., 1995, 36:229-240; Richieri, G. V. et al., J. Biol. Chem., 1992, 267:23485-23501).
Current diagnostic methods do not measure serum FFAU levels but, instead, monitor FFA metabolism relative to abnormal cardiac conditions. These methods generally involve injecting radioactive compounds which are then detected in the patient using tomography imaging (Franken, P. R. et al., Acta Cardiol., 1996, 51(6):501-514; Takeishi, Y. et al., Nucl. Med. Commun., 1996, 17(8):675-680; Shimonagata, T. et al., Diabetes Care, 1998, 19(8):887-891; Indolfi C. et al., Am. Heart J., 1996, 132(3):542-549; Kondo, C. et al., J. Nucl. Cardiol., 1996, 3(1):30-36; Chen S. L. et al., 1995, Nucl. Med. Commun. 16(5):336-343; Nozaki T. et al., 1995, J. Nucl. Med. 36(3):518-524). The fatty acid analogs frequently used include 123I-BMIPP and xcex2-methyl[1xc2x714C]heptadecanoic acid and the methods of detection include positron emission tomography (PET) or single-proton-emission computed tomography (SPECT) (Franken, P. R. et al., Acta Cardiol., 1996, 51(6):501-514; Knuuti M. J. et al., J. Mol. Cell. Cardiol., 1995, 27(7):1359-1367; Nozaki T. et al., 1995, J. Nucl. Med. 36(3):518-524). These methods are relatively complex and costly because of the equipment required for testing. Thus, there is a need for a simple test for determining FFA in serum to reliable diagnosis of cardiac ischemia.
Drugs for lowering plasma levels of FFA or treating ischemic conditions are known (U.S. Pat. No. 5,589,467; U.S. Pat. No. 5,484,774; U.S. Pat. No. 5,430,027; U.S. Pat. No. 5,032,583). Drugs with such activities are potentially useful for treating hyperlipidemias, brain ischemia and cardiovascular disorders such as cardiac ischemia, cardiac arrhythmias, angina, hypertension and heart failure. Thus, there is a need for an assay for determining the efficacy of known drugs for lowering levels of serum FFA in a patient and for discovery of drugs with this property.
The present invention is a relatively simple method of detecting FFAU levels in serum that are associated with and diagnostic of cardiac ischemic conditions. The present invention is useful as a diagnostic tool for detecting cardiac ischemia, alone or in conjunction with other diagnostic determinations. It is also useful for monitoring the efficacy of treatments of cardiac disorders and for discovery of new agents that modulate serum levels of FFA.
According to the invention, there is provided a method of detecting cardiac ischemia in a mammal, comprising the steps of providing a serum sample obtained from the mammal, mixing the serum sample with an aqueous solution and with a reagent comprising a fatty acid binding protein labeled with a fluorescent moiety, wherein the reagent exhibits a first fluorescence in an aqueous solution and a measurably different second fluorescence in an aqueous solution when the fatty acid binding protein is bound to a free fatty acid, wherein the free fatty acid is unbound to serum albumin, measuring the second fluorescence after the serum sample is mixed with the aqueous solution and the reagent to determine a concentration of the free fatty acid in the serum sample, and determining whether the concentration of the free fatty acid in the serum sample is indicative of cardiac ischemia. In one embodiment, the providing step comprises providing a serum sample from a human. In another embodiment, the measuring step of the second fluorescence is performed at a wavelength that differs from a wavelength at which the reagent exhibits the first fluorescence. Preferably, the wavelength for measuring the second fluorescence is about 430 nm to about 450 nm, and the wavelength at which the reagent exhibits the first fluorescence is about 500 nm to about 550 mm. In one embodiment of the method, the determining step comprises determining that the concentration of the free fatty acid unbound to serum albumin in the serum sample is significantly higher than a concentration of free fatty acid unbound to serum albumin in serum of a control population that does not have cardiac ischemia. In another embodiment, the determining step comprises determining that the concentration of the free fatty acid unbound to serum albumin in the serum sample is about two standard deviations greater than an average concentration of free fatty acid unbound to serum albumin in serum of a control population that does not have cardiac ischemia. In a preferred embodiment, the determining step further comprises determining a degree of cardiac ischemia by determining that the concentration of the free fatty acid unbound to serum albumin in the serum sample is at least about two-fold greater than an average concentration of free fatty acid unbound to serum albumin in serum of the control population. In one embodiment, the determining step comprises determining that the concentration of the free fatty acid unbound to serum albumin in the serum sample is at least about 12 nM. Another embodiment of the method further comprises the steps of measuring total free fatty acid and albumin in the serum sample, and determining a ratio of the total free fatty acid and the albumin. In another embodiment, the reagent in the mixing and measuring steps is a fatty acid binding protein labeled with a fluorescent moiety, the fatty acid binding protein is a rat intestinal fatty acid binding protein, a human adipocyte fatty acid binding protein, or a human heart fatty acid binding protein, and the fluorescent moiety is acrylodan, danzyl aziridine, 4-[N-[(2-iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole ester (IANBDE), or 4-[N-[(2-iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBDA). In a preferred embodiment, the reagent in the mixing and measuring steps is a fatty acid binding protein labeled with acrylodan, the fatty acid binding protein is a mutant protein comprising a rat intestinal fatty acid binding protein having a cysteine at residue 27, 81, 82, or 84, or an alanine at residue 72, or a human heart fatty acid binding protein having a lysine at residue 27. In another preferred embodiment, the reagent in the mixing and measuring steps is a rat intestinal fatty acid binding protein labeled with acrylodan. In another embodiment, the reagent in the mixing and measuring steps is a rat intestinal fatty acid binding protein labeled with a fluorescent moiety, wherein the fatty acid binding protein comprises a site-specific mutant in which at least one amino acid residue has been altered. Preferably, the reagent is a rat intestinal fatty acid binding protein labeled with acrylodan and having an alanine at residue 72.